The present invention relates to methods for treating various benign or malignant neoplasms, chronic infections, autoimmune diseases, and immunodeficiencies. In particular, the present invention relates to methods of treating the growth and metastasis of various malignancies with a botulinum toxin.
Neoplasms
The initial growth of neoplasms is dependent upon adequate supply of growth factors and the removal of toxic molecules. The expansion of tumor mass beyond 2 mm in diameter depends on the development of angiogenesis to produce adequate blood supply. The induction of angiogenesis is mediated by multiple molecules that are released by both tumor cells and host cells, including endothelial cells, epithelial cells, mesothelial cells, and leukocytes. Angiogenesis consists of sequential processes emanating from microvascular endothelial cells. As it expands, the tumor (primary or secondary) can also cause certain symptoms, such as discomfort (e.g., the feeling of a lump), pain and bleeding.
After angiogenesis begins, tumor cell invasion of the tissue surrounding the primary tumor and penetration of blood and lymph vessels is central to the whole phenomenon of metastasis.
Once tumor cells detach from the primary tumor, they must invade the host stroma to penetrate lymphatics and blood vessels. To do so, tumor cells must penetrate basement membranes surrounding blood vessels. Basement membranes and connective tissue extracellular matrix (ECM) consist of four major groups of molecules: collagens, elastins, glycoproteins, and proteoglycans. The degradation of the ECM and basement membrane components by tumor cells is an essential prerequisite for invasion and metastasis.
In sum, cancer metastasis consists of multiple complex, interacting, and interdependent steps, each of which is rate-limiting, since a failure to complete any of the steps prevents the tumor cell from producing a metastasis. The tumor cells that eventually give rise to metastases must survive a series of potentially lethal interactions with host homeostatic mechanisms. The balance of these interactions can vary among different patients with different neoplasms or even among different patients with the same type of neoplasm.
The essential steps in the formation of a metastasis are similar in all tumors and consist of the following:                1. Alter neoplastic transformation, progressive proliferation of neoplastic cells is initially supported with nutrients supplied from the organ microenvironment by diffusion.        2. Neovascularization or angiogenesis must take place for a tumor mass to exceed 1 or 2 mm in diameter. The synthesis and secretion of different angiogenic molecules and suppression of inhibitory molecules are responsible for the establishment of a capillary network from the surrounding host tissue.        3. Some tumor cells can down regulate expression of cohesive molecules and have increased motility and, thus, can detach from the primary lesion. Invasion of the host stroma by some tumor cells occurs by several parallel mechanisms. Capillaries and thin-walled venules, like lymphatic channels, offer very little resistance to penetration by tumor cells and provide the most common pathways for tumor cell entry into the circulation.        4. Detachment and embolization of single tumor cells or cell aggregates occur next, the vast majority of circulating tumor cells being rapidly destroyed.        5. Once the tumor cells have survived the circulation, they must . . . .        6. Arrest in the capillary beds of distant organs by adhering either to capillary endothelial cells or to exposed subendothelial basement membranes.        7. Tumor cells (especially those in aggregates) can proliferate within the lumen of the blood vessel, but the majority extravasate into the organ parenchyma by mechanisms similar to those operative during invasion.        8. Tumor cells bearing appropriate cell surface receptors can respond to paracrine growth factors and hence proliferate in the organ parenchyma.        9. The metastatic cells must evade destruction by host defenses that include specific and nonspecific immune responses.        10. To exceed a mass of 1 to 2 mm in diameter, metastasis must develop a vascular network.Botulinum Toxin        
The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin. To date seven immunologically distinct botulinum neurotoxins have been characterized: serotypes A, B, C1, D, E, F, and G. Of these Botulinum toxin type A is recognized as one of the most lethal naturally occurring agents known to man.
It is postulated that the botulinum toxins bind with high affinity to cholinergic motor neurons, are transferred into the neuron and effectuate blockade of the presynaptic release of acetylcholine. All of the botulinum toxin serotypes are purported to inhibit release acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. For example, Botulinum toxin A is a zinc endopeptidase which can specifically hydrolyze a peptide linkage of the intracellular, vesicle associated protein SNAP-25. Botulinum type E also cleaves the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), however, type E binds to a different amino acid sequence within SNAP-25. It is believed that differences in the site of inhibition is responsible for the relative potency and/or duration of action of the various botulinum toxin serotypes.
Currently, Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles. Botulinum toxin type A was approved by the U.S. Food and Drug Administration in 1989 for the treatment of essential blepharospasm, strabismus and hemifacial spasm in patients over the age of twelve. In 2000, the FDA approved commercial preparations of type A and type B botulinum toxin serotypes for the treatment of cervical dystonia, and in 2002 the FDA approved a type A botulinum toxin for the cosmetic treatment of certain hyperkinetic (glabellar) facial wrinkles. In 2004, the FDA approved botulinum for the treatment of hyperhidrosis. Non-FDA approved uses hemifacial spasm, spasmodic torticollis, oromandibular dystonia, spasmodic dysphonia and other dystonias, tremor, myofascial pain, temporomandibular joint dysfunction, migraine, spasticity.
Clinical effects of peripheral intramuscular botulinum toxin type A are usually seen within 24-48 hours of injection and sometimes within a few hours. When used to induce muscle paralysis, symptomatic relief from a single intramuscular injection of botulinum toxin type A can last approximately three months, however, under certain circumstances effects have been known to last for several years.
Despite the apparent difference in serotype binding, it is thought that the mechanism of botulinum activity is similar and involves at least three steps. First, the toxin binds to the presynaptic membrane of a target cell. Second, the toxin enters the plasma membrane of the effected cell wherein an endosome is formed. The toxin is then translocated through the endosomal membrane into the cytosol. Third, the botulinum toxin appears to reduce a SNAP disulfide bond resulting in disruption in zinc (Zn++) endopeptidase activity, which selectively cleaves proteins essential for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane. Tetanus neurotoxin, botulinum toxin B, D, F, and G cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytosolic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. Each toxin specifically cleaves a different bond.
The molecular weight of the botulinum toxin protein molecule, for all seven of the known botulinum toxin serotypes, is about 150 kD. Interestingly, the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, the botulinum toxin type A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. Botulinum toxin types B and C.sub.1 are apparently produced as only a 500 kD complex. Botulinum toxin type D is produced as both 300 kD and 500 kD complexes. Finally, botulinum toxin types E and F are produced as only approximately 300 k) complexes. The complexes (i.e. molecular weight greater than about 150 kD) are believed to contain a non-toxin hemaglutinin protein and a non-toxin and non-toxic nonhemaglutinin protein. These two non-toxin proteins (which along with the botulinum toxin molecule can comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when toxin is ingested. Additionally, it is possible that the larger (greater than about 150 kD molecular weight) botulinum toxin complexes may result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex. The toxin complexes can be dissociated into toxin protein and hemagglutinin proteins by treating the complex with red blood cells at pH 7.3. The toxin protein has a marked instability upon removal of the hemagglutinin protein.
All the botulinum toxin serotypes are made by Clostridium botulinum bacteria as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C.sub.1, D, and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture. Serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form. However, even the proteolytic strains that produce, for example, the botulinum toxin type B serotype only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of, for example, the botulinum toxin type B toxin is likely to be inactive, possibly accounting for a lower potency of botulinum toxin type B as compared to botulinum toxin type A. The presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the overall protein load of the preparation, which has been linked to increased antigenicity, without contributing to its clinical efficacy.
In vitro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and glutamate.
High quality crystalline botulinum toxin type A can be produced from the Hall A strain of Clostridium botulinum with characteristics of 3.times.10.sup.7 U/mg, an A.sub.260/A.sub.278 of less than 0.60 and a distinct pattern of banding on gel electrophoresis. The known Shantz process can be used to obtain crystalline botulinum toxin type A, as set forth in Shantz, E. J., et al, Properties and use of Botulinum toxin and Other Microbial Neurotoxins in Medicine, Microbiol Rev. 56: 80-99 (1992). Generally, the botulinum toxin type A complex can be isolated and purified from an anaerobic fermentation by cultivating Clostridium botulinum type A in a suitable medium. Raw toxin can be harvested by precipitation with sulfuric acid and concentrated by ultramicrofiltration. Purification can be carried out by dissolving the acid precipitate in calcium chloride. The toxin can then be precipitated with cold ethanol. The precipitate can be dissolved in sodium phosphate buffer and centrifuged. Upon drying there can then be obtained approximately 900 kD crystalline botulinum toxin type A complex with a specific potency of 3.times.10.sup.7 LD.sub.50 U/mg or greater. This known process can also be used, upon separation out of the non-toxin proteins, to obtain pure botulinum toxins, such as for example: purified botulinum toxin type A with an approximately 150 kD molecular weight with a specific potency of 1-2.times.10.sup.8 LD.sub.50 U/mg or greater; purified botulinum toxin type B with an approximately 156 kD molecular weight with a specific potency of 1-2.times.10.sup.8 LD.sub.50 U/mg or greater, and; purified botulinum toxin type F with an approximately 155 kD) molecular weight with a specific potency of 1-2.times.10.sup.7 LD.sub.50 U/mg or greater.
Already prepared and purified botulinum toxins and toxin complexes suitable for preparing pharmaceutical formulations can be obtained from List Biological Laboratories, Inc., Campbell, Calif.; the Centre for Applied Microbiology and Research, Porton Down, U.K.; Wako (Osaka, Japan), as well as from Sigma Chemicals of St Louis, Mo.
The pattern of toxin spread within a muscle has been demonstrated to be related to concentration, volume and location of injection site (22).
Prior Art Using a Neurotoxin to Treat Cancer
Several patents and applications have taught treating cancers with a neurotoxin and specifically a botulinum toxin. Uniformly, the methods have taught to directly deliver botulinum toxin to the cancerous cells or their vicinity with the goal of directly affecting the cancerous cells or their innervation. The goal has been to deliver the toxin into the cancerous cell to exert an effect, or to locally denervate a cancerous cell. By getting the toxin into a cell, botulinum toxin may inhibit the process of exocytosis from the cancer cell, which is the release of a cell's intracellular contents or vesicles into the extracellular space. These patents and applications teach, in part, that inhibiting exocytosis of a cancer cell will reduce the activity of a cells division and reduce the ability of the cancer cell to move. By locally denervating a cancer cell, it may become less active. The following review of prior art will address these issues.
Patent application US2005/0031648 A1, Methods for Treating Diverse Cancers, discloses the treatment of hyperplastic, precancerous or cancerous tissues with a botulinum neurotoxin by locally administering the botulinum toxin to the hyperplastic, precancerous or cancerous tissue or to its vicinity. Only diseased tissue is “treated.” Local administration is defined as direct injection of the neurotoxin into or to the local area of the target tissue (para 178). “Treatment” is defined as specifically causing the reduction in the size and or activity of a hyperplastic, hypertonic or neoplastic tissue, by targeting the cancer cells directly or the “vicinity of the target tissue” (para 159). This patent application further states that a “therapeutically effective amount” will not cause significant negative or adverse side effects to the treated tissue (para 177). The patent application acknowledges that in order to achieve the desired effect in a non-neuronal cell (such as a breast cancer) the injected dose may need to be higher (0187) since non-nerve cells do not contain cell surface receptors for a botulinum toxin. In fact, such cells must be permeabilized to allow entry in vitro of the botulinum toxin into the cells cytosol (para 191). In addition, this patent application states that a botulinum toxin can block the release of any vesicle mediated endocytosis as long as the light chain of the botulinum toxin is translocated into the intracellular medium (para 191).
Patent application WO 2005/030248 attempts to overcome the significant shortfall of botulinum toxin when directly treating cancer cells, namely that the toxin does not have a high affinity for non-neuronal cells and therefore much higher doses are needed to enter such a cell. Once the toxin is into a cell, the toxin will interfere with the intracellular machinery. This application describes a method of increasing the entry of botulinum toxin c3 into cancer cells by linking the c3 to a cell-permeable fusion protein. The goal of the treatment is to stop the cancer cell from contracting and spreading. The described compound specifically targets a cancer cell. This patent application teaches that the compound can be injected around a cancer at the ‘resection margin’ following surgery as well, but the goal is to treat cancerous cells that may reside at that margin. The method of injection a resection margin necessarily implies that surgery has been performed to achieve a ‘resection margin’ that can be treated.
US 2002/0094339 A1, U.S. Pat. Nos. 6,565,870 B1 and 6,139,845 all teach treating tumors, cancers and disorders with a botulinum toxin. The toxin is injected directly into the diseased tissue to exert its effect in inhibiting exocytosis.
Limitations of Prior Art
The prior art relies on the delivery of botulinum toxin directly into the cancer cells or to their immediate local vicinity to achieve an effect on the cancer cells. There are several substantial shortcomings of these methods which, if employed, may cause significant negative or adverse side effects to the treated tissue or its surroundings.
The first limitation of the prior art relates to the cholinergic innervation of cancer. The scientific literature is replete with reports that cancers are cholinergically innervated and that blocking this cholinergic innervation (with botulinum toxin) may reduce the ability of the cancer cell to divide, spread or invade locally. However, it is also clear that some cancers have the opposite effect by cholinergic stimulation, and that blocking this cholinergic innervation (with botulinum toxin) may actually enhance the ability of the cancer cell to divide, spread or invade locally, In fact, there are conflicting scientific reports that the same type of cancer (lung) may be stimulated or inhibited by cholinergic stimulation. When one considers the well-known fact that cancers are not a homogeneous population of cells but are usually a heterogeneous mixture, or that cancer cells are capable of modifying their responses, it becomes clear that blocking cholinergic innervation of a cancer may be good for some parts of a cancer, but bad for other parts. Alternatively a cancer that shrinks today from blocking cholinergic innervation may adapt and become stimulated by cholinergic innervation tomorrow.
A second significant limitation of the prior art relates to the fundamental concept of getting botulinum toxin into a cancer cell, where a cell's entire capacity to undergo exocytosis can be affected. US 2005/0031648 teaches that the substrate for a botulinum toxin is not restricted to neuronal cells which release acetylcholine, rather the substrates are “ubiquitously involved in membrane-membrane fusion events” and evidence points to “a universal mechanism for membrane fusion events” (para 0103). Once the toxin has been internalized into the affected cell, it acts in the known manner, as an endoprotease upon its respective secretory vessel-membrane docking protein (para 187). Furthermore, “as the concentration is raised, non-cholinergic sympathetic neurons, chromaffin cells and other cell types can take up a botulinum toxin and show reduced exocytosis” (para 191). Again, the goal is to reduce the excessive secretions from some cancerous cell by interfering with exocytosis, as also indicated in US 2002/0094339 A1, U.S. Pat. Nos. 6,139,845 and 6,565,870.
One fundamental problem of delivering the toxin into the cancer cell or diseased tissue's cell to interfere with exocytosis is that exocytosis within cancer cells is an absolute necessity to help kill cancer cells. Any attempt to globally eliminate or reduce exocytosis could certainly be hazardous, since all cells (including cancer cells) routinely undergo processing and presentation of molecules to their surface through exocytosis which serve to signal to the body's immune system whether a cell is cancerous or normal. Depending on the type of molecule presented to its surface, a cell may be killed by the immune system if it is considered ‘cancerous’ or protected if considered normal or ‘self’. The process of cell signaling is dependent on an intact process of exocytosis. If these so-called tumor associated antigens are eliminated from the surface of a cancer cell by globally inhibiting exocytosis from within a cancer cell, the immune system will be less likely, if at all, to destroy the foreign cancer cell. Injecting directly into a cancer cell would further increase significant negative or adverse side effects of the treated tissue.
Thirdly, it has been taught that non-neuronal cells are less sensitive to botulinum toxin (US 2005/0031648 at para 191) and that, “as the toxin concentration is raised, non-cholinergic sympathetic neurons, chromaffin cells and other cell types can take up a botulinum toxin and show reduced exocytosis.” It is well known to one skilled in the art that injections of higher concentrations of toxin are associated with a higher incidence of local side effects, because of spread to surrounding muscles and inadvertent muscle paralysis. Therefore, a high dose or concentration of toxin that is required to enter non-neuronal cells may cause excessive spread from the target area and cause affect not only the cancer cells, but also a significant area of surrounding tissue causing unacceptable side effects.
Fourth, there are other practical limitations of directly injecting a cancer or its vicinity with any substance, including botulinum. By directly injecting a cancer, one must insert a needle directly into to cancer and inject under pressure the desired substance. The needle may pass into then through the cancerous tissue and possibly seed cancerous cells into an area that did not contain cancerous cells in the first place. Even if the needle does not pass through the cancer, the pressure effect of the injectate may force or push cancerous cells into the surrounding normal tissue or into the thin walled lymphatics or blood vessels within or in the vicinity of the cancer, causing a higher chance of significant negative or adverse side effects, namely regional or distant metastases. For example, it is well known that one may bleed temporarily when a needle is stuck into the skin or gum following a dental visit. When this needle is entering a cancer, one must consider that the blood vessels have been broken and, at the microscopic level, cancer cells may have entered the circulation.
Fifth, patent application WO 2005/030248 describes a method of reducing actin filament association by getting botulinum toxin into cancer cells. Although it is taught that this may prevent cancer cells from contracting and migrating, it may also lead to loss of adhesion of malignant cells and result in increased distant spread.
Accordingly, there is a need for an effective method of treating a cancer using a neurotoxin. There is also a need for using a neurotoxin to treat other conditions.